This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy of inherited diseases affecting the function of human phagocytic cells, including neutrophils,eosinophils, and monocytes. A related goal of this project is development of novel cellular therapies based on genetically engineering human PBHP and their phagocytic cell progeny to endow them with new properties to augment host defense against chronic infections with intracellular pathogens including tuberculosis and other mycobacteria infections. We have defined conditions for optimum harvest, purification and culture of the primitive human hematopoietic cells with a CD34 surface antigen phenotype. In a clinical study we determined that 10 ug/kg daily subcutaneous granulocyte colony stimulating factor (G-CSF) administered for 5 or 6 daily doses optimizes recruitment of circulating primitive CD34 progenitor cells, and that recruitment is transient, peaking on day 5. Using a antibody- magnetic bead technology developed by Baxter Healthcare we have achieved purification of 50 to 200 million CD34+ cells from donors, and used these cells for development of gene transfer therapy. In related studies we have studied optimum conditions for growth of CD34+ cells in culture using stem cell factor, IL3, IL6, plus the additional early acting factor, FLT3 resulting in expansion of very early progenitors capable of generating GEMM colonies. We have developed specific culture conditions leading to predominantly neutrophils, eosinophils or monocyte/macrophages. This will be important for studies aimed at engineering new characteristics into specific end stage phagocytes by gene transfer into progenitors. Using conditions for monocyte differentiation we introduced a gene for interferon gamma (IFN-gamma) into these cells by targeting the CD4+ progenitors, finding that this resulted in augmentation of oxidase activity and an increase in IgG Fc receptors. In other studies, the gene for nitric oxide synthase (NOS) was successfully transferred into hematopoietic progenitor cell lines. This enzyme produces nitric oxide, an oxidant implicated in host defense against intracellular pathogens, including tuberculosis. These studies of IFN-gamma or NOS gene transfer into hematopoietic progenitors has potential as a means of augmenting monocyte host defense activities for treatment of chronic intracellular infections.